Antimicrobial activity of enacyloxin IIa and gladiolin against the urogenital pathogens Neisseria gonorrhoeae and Ureaplasma spp

Aims To determine the antimicrobial activity of enacyloxin IIa and gladiolin against N. gonorrhoeae and Ureaplasma spp. Methods and Results The Burkholderia polyketide antibiotics enacyloxin IIa and gladiolin were tested against 14 N. gonorrhoeae and 10 Ureaplasma spp. isolates including multi‐drug resistant N. gonorrhoeae isolates WHO V, WHO X and WHO Z as well as macrolide, tetracycline and ciprofloxacin resistant ureaplasmas. Susceptibility testing of N. gonorrhoeae were carried out by agar dilution, whereas broth micro‐dilution and growth kinetic assays was used for Ureaplasma spp. The MIC range for enacyloxin IIa and gladiolin against N. gonorrhoeae was 0.015 – 0.06 mg/L and 1 ‐ 2 mg/L, respectively. The presence of resistance to front line antibiotics had no effect on MIC values. The MIC range for enacyloxin IIa against Ureaplasma spp. was 4 – 32 mg/L with a clear dose‐dependent effect when observed using a growth kinetic assay. Gladiolin had no antimicrobial activity on Ureaplasma spp. at 32 mg/L and limited impact on growth kinetics. Conclusions Enacyloxin IIa and gladiolin antibiotics have antimicrobial activity against a range of antibiotic susceptible and resistant N. gonorrhoeae and Ureaplasma isolates. Significance and Impact of Study This study highlights the potential for a new class of antimicrobial against pathogens in which limited antibiotics are available. Development of these compounds warrants further investigation in the face of emerging extensively‐drug resistant strains

Identification of common genetic variation that modulates alternative splicing

Alternative splicing of genes is an efficient means of generating variation in protein function. Several disease states have been associated with rare genetic variants that affect splicing patterns. Conversely, splicing efficiency of some genes is known to vary between individuals without apparent ill effects. What is not clear is whether commonly observed phenotypic variation in splicing patterns, and hence potential variation in protein function, is to a significant extent determined by naturally occurring DNA sequence variation and in particular by single nucleotide polymorphisms (SNPs). In this study, we surveyed the splicing patterns of 250 exons in 22 individuals who had been previously genotyped by the International HapMap Project. We identified 70 simple cassette exon alternative splicing events in our experimental system; for six of these, we detected consistent differences in splicing pattern between individuals, with a highly significant association between splice phenotype and neighbouring SNPs. Remarkably, for five out of six of these events, the strongest correlation was found with the SNP closest to the intron-exon boundary, although the distance between these SNPs and the intron-exon boundary ranged from 2 bp to greater than 1,000 bp. Two of these SNPs were further investigated using a minigene splicing system, and in each case the SNPs were found to exert cis-acting effects on exon splicing efficiency in vitro. The functional consequences of these SNPs could not be predicted using bioinformatic algorithms. Our findings suggest that phenotypic variation in splicing patterns is determined by the presence of SNPs within flanking introns or exons. Effects on splicing may represent an important mechanism by which SNPs influence gene function

Manuka honey inhibits the development of Streptococcus pyogenes biofilms and causes reduced expression of two fibronectin binding proteins

Streptococcus pyogenes (group A Streptococcus; GAS) is always of clinical significance in wounds where it can initiate infection, destroy skin grafts and persist as a biofilm. Manuka honey has broad spectrum antimicrobial activity and its use in the clinical setting is beginning to gain acceptance with the continuing emergence of antibiotic resistance and the inadequacy of established systemic therapies; novel inhibitors may affect clinical practice. In this study, the effect of manuka honey on S. pyogenes (M28) was investigated in vitro with planktonic and biofilm cultures using MIC, MBC, microscopy and aggregation efficiency. Bactericidal effects were found in both planktonic cultures and biofilms, although higher concentrations of manuka honey were needed to inhibit biofilms. Abrogation of adherence and intercellular aggregation was observed. Manuka honey permeated 24 h established biofilms of S. pyogenes, resulting in significant cell death and dissociation of cells from the biofilm. Sublethal concentrations of manuka honey effectively prevented the binding of S. pyogenes to the human tissue protein fibronectin, but did not inhibit binding to fibrinogen. The observed inhibition of fibronectin binding was confirmed by a reduction in the expression of genes encoding two major fibronectin-binding streptococcal surface proteins, Sof and SfbI. These findings indicate that manuka honey has potential in the topical treatment of wounds containing S. pyogenes. INTRODUCTION Streptococcus pyogenes (group A Streptococcus) colonizes the nasopharynx and skin of healthy individuals, forming part of the commensal microbiota. Under appropriate conditions, S. pyogenes can be transmitted to wounds and is especially problematic after surgery, following skin grafting and for military personal with traumatic or puncture wounds. Wounds provide a route of entry to the host and damaged tissues display a matrix of proteins including collagen, albumin, fibronectin and fibrinogen, which collectively provide a plethora of ligands to which opportunistic pathogens, including streptococci, adhere Biofilms have been associated with persistent or chronic wound infections and are a major obstacle to healing Honey has had a valued place in traditional medicine for many centuries and was reintroduced into modern medicine during the 21st century. Honey exhibits broad spectrum antibacterial activities and has been reported to inhibit more than 80 species of bacteria, including meticillin-resistant Staphylococcus aureus, vancomycin-resistant enterococci, Lancefield groups A, C and G streptococci, Pseudomonas aeruginosa and Actinomyces species METHODS Bacterial strains. S. pyogenes MGAS6180 (M28, invasive disease; Green et al., 2005) was grown in Todd-Hewitt (TH) broth (Oxoid) containing 0.5 % yeast extract; Bacto agar (Difco) was added to achieve a 1.4 % (w/v) final concentration in agar plates. Cultures were incubated at 37 uC in air supplemented with 5 % CO 2 . For analysis of aggregation properties, S. pyogenes was grown in C medium containing 0.2 % glucose (Lyon et al., 1998). Medical grade manuka honey. Sterile medical grade manuka honey (Medihoney) was kindly donated by Comvita. Medihoney was provided in sterile 50 g portions. Minimum inhibitory and bactericidal concentrations. The MIC for manuka honey against planktonically grown S. pyogenes MGAS6180 was determined by serial dilution (0-95 %; w/v) in a total volume of 5 ml iso-sensitest broth (Oxoid) (according to the British Society for Antimicrobial Chemotherapy methodology for determining MIC; Andrews, 2011). Cultures were incubated for 16 h at 37 uC in aerobic conditions with 5 % CO 2 . To establish the MBC, samples corresponding to the MIC and including three samples of a higher concentration were plated onto Todd-Hewitt agar and incubated for 16 h at 37 uC in aerobic conditions with 5 % CO 2 . Assays were done in triplicate on each of three separate occasions. Inhibition of bacterial growth. To determine the extent of growth inhibition by manuka honey, triplicate cultures of S. pyogenes MGAS6180 were grown for 8 h at 37 uC in 10 ml TH broth, in aerobic conditions with 5 % CO 2 , supplemented with manuka honey (0, 20 and 40 % w/v). Bacterial growth was monitored at OD 650 at hourly intervals. Growth experiments also utilized triplicate biological samples. Aggregation assays. S. pyogenes MGAS6180 was initially grown in C medium for 16 h (Lyon et al., 1998) and harvested by centrifugation at 5000 g for 10 min. Bacterial cell pellets were resuspended in either 1 ml C medium or an appropriate concentration of manuka honey (5 and 10 %; w/v) dissolved in C medium and the OD 600 of the culture was adjusted to 1.0 (±0.05) if necessary. In both cases, manuka honey at twice the desired concentration was dissolved in double strength TH and diluted to the required concentration using TH; cell pellets were resuspended in either 5 or 10 % (w/v) honey solutions. In an untreated control, manuka honey was replaced with PBS added to maintain the appropriate volume and concentration of TH media. Aggregation assays were carried out in triplicate as described previously Static biofilm model. S. pyogenes MGAS6180 was initially grown in C medium for 16 h and these stationary phase cultures were harvested by centrifugation and adjusted to OD 650 0.1. To determine whether manuka honey prevented biofilm formation, biofilms were established in 96-well microtitre plates (Greiner) in 50 ml TH, supplemented with manuka honey (0, 10, 20 and 40 % w/v), by inoculating each well with 5 ml harvested cells. Plates were incubated at 37 uC for 24 h, aerobically with 5 % CO 2 . To estimate biomass, unattached cells were gently aspirated and discarded, and adherent cells were washed twice with PBS and stained with crystal violet (0.25 %; w/v) for 10 min; following a further two washes with PBS, cell-bound crystal violet was resolubilized with 7 % acetic acid, and absorbance was measured at 595 nm (A 595 ) Live-Dead staining. Images of bacterial cells were collected for control cells (untreated) and for cells treated with 40 % (w/v) manuka honey for 2 h, to determine the effect of manuka honey on viability. Biofilms were grown in Petri dishes in 5 ml TH media, or 5 ml 40 % (w/v) honey dissolved in TH media, as previously described; liquid was aspirated from the plates and biofilms were washed with 1 ml PBS. Biofilms were scraped from the coverslips using a cell scraper, resuspended in 1 ml PBS and stained with Live-Dead BacLight bacterial viability kit (Invitrogen), following the manufacturer's instructions. Fluorescence microscopy images were obtained using a Nikon Eclipse 80i fluorescent microscope with oil immersion and 6100 lens. For detection of SYTO 9 (green channel) a 488 nm excitation and 520 nm emission filter was used. For propidium iodide detection (red channel) a 543 nm excitation and 572 nm emission filter was used. Image analysis used Volocity Software (PerkinElmer). Biofilm disruption. To determine whether manuka honey affected biofilm biomass by facilitating the dissociation of adherent cells from S. E. Maddocks and others 782 Microbiology 158 the biofilm, assays were conducted to monitor the numbers of viable planktonic cells that were released into the liquid phase, from established biofilms following treatment with manuka honey. Biofilms were grown for 24 h as described above. The liquid was aspirated from each well, biofilms were washed twice with PBS to remove any planktonic or loosely adherent cells, and manuka honey over a range of concentrations [0, 10, 20 and 40 % (w/v), respectively] was added to the 24 h established biofilms. Following the application of honey, biofilms were incubated for a further 2 h at 37 uC as above and samples of the liquid above the biofilm were collected at 30 min intervals. Bacterial cells were enumerated using the total viable cell (TVC) counting method described by 21 . Fibronectin-and fibrinogen-binding assays. To determine the effect of manuka honey on adherence of S. pyogenes cells to immobilized fibronectin and fibrinogen, a crystal violet assay was conducted as described previously, using 1 % BSA to block wells prior to assaying cell binding RNA extraction from S. pyogenes biofilms. Large scale, static biofilms of S. pyogenes were grown in duplicate in 5 ml C medium (with 20 % honey for the 'treated' biofilms) in sterile Petri dishes for 24 h at 37 uC, as for the small scale biofilms. The liquid was aspirated and the biofilm was scraped from the surface of the Petri dish using a sterile cell scraper. Biofilms were resuspended in 500 ml PBS and vortexed for 1 min to break up cell aggregates. Honey-treated and untreated cell suspensions were equilibrated (to approximately 2.5610 9 c.f.u.) prior to treatment with mutanolysin (100 mg) and lysozyme (100 mg) for 20 min at 37 uC. RNA extraction was carried out using the SV Wizard total RNA extraction kit (Promega) according the manufacturer's instructions. RNA quantification was performed by spectrophotometric measurement using a NanoDrop ND-1000 (NanoDrop Technologies) and each RNA sample was adjusted to give a final concentration of 10 ng ml 21 . End point RT-PCR to determine the relative expression of sof and sfbI. PCR primers were designed to amplify a 1100 bp fragment of the sof gene (sof-fwd: 59-ACTTAGAAAGTTATCTGTAGGG; sofrev: 59-TCTCTCGAGCTTTATGGATAG) and 1200 bp fragment of the sfbI gene (sbfI-fwd: 59-AACTGCTTTAGGAACAGCTTC; sbfI-rev: 59-CCACCATAGCCACAATGCT). The complete genome sequence for S. pyogenes MGAS6180 was obtained from the NCBI database (www.ncbi.nlm.nih.gov) and used as a basis to design the primers used in this study. Internal control primers were designed to amplify a 900 bp internal fragment of the glr (murI; glutamate racemase) gene (glr-fwd: 59-ATGGATACAAGACCAATTGGG; glr-rev: 59-TCATAA-GGTGACATGCTCCAC), a known housekeeping gene in S. pyogenes that is commonly used for MLST (http://spyogenes.mlst.net/misc/ info.asp) RESULTS Inhibition of planktonic S. pyogenes by manuka honey The MIC of manuka honey against S. pyogenes MGAS6180 was found to be 20 % (w/v) and the MBC was found to be 45 % (w/v). Growth curves with 20 % (w/v) manuka honey resulted in a reduced growth rate and reduction in overall cell number ( S. pyogenes aggregation and biofilm development are inhibited by sublethal concentrations of manuka honey To test the capacity for manuka honey to inhibit intercellular aggregation, suspensions of S. pyogenes MGAS6180 were mixed with 10 % (w/v) manuka honey. In the absence of manuka honey, cells strongly aggregated but in the presence of 10 % (w/v) honey, aggregation was completely inhibited (P,0.05) When biofilms of S. pyogenes were initiated in the presence of 10, 20 and 40 % (w/v) manuka honey, a statistically significant reduction in biomass was observed in each case. A reduction of 75 % (P50.03) was observed with 10 % (w/ v) manuka honey; 20 and 40 % (w/v) manuka honey resulted in between 90 % (P50.02) and 96 % (P50.01) reduction in biomass, respectively Manuka honey facilitates cell death and dissociation of bacterial cells from established S. pyogenes biofilms To determine the effect of manuka honey against established biofilms of S. pyogenes MGAS6180, biofilms were grown for 24 h prior to honey treatment. Following 2 h treatment with 10 and 20 % manuka honey (w/v), a 72 % (P50.32) and 77 % (P50.08) reduction in biomass was observed, respectively. Following treatment for 2 h with 40 % (w/v) manuka honey, the observed reduction in biofilm biomass was even greater, and statistically significant at 85 % (P50.01) Live-Dead viability staining (Invitrogen) of untreated biofilms grown for 24 h at 37 uC, honey-treated (40 % w/ v) biofilms grown for 24 h at 37 uC, and biofilms that were grown for 24 h at 37 uC, then treated for 2 h with 40 % (w/ v) manuka honey, showed that in both cases, the honey treatment resulted in cell death With time, bacterial cells may dissociate from a biofilm. To determine whether manuka honey facilitated the dissociation of S. pyogenes from biofilms, the levels of planktonic cells in the liquid phase were monitored for 2 h following the application of manuka honey (the biofilm data described above showed that the biomass was reduced following 2 h treatment, so this time period was deemed appropriate for these experiments). Immediately after application of the manuka honey (0 min) the c.f.u. was approximately the same for each condition. After 30 min and throughout the duration of the experiment (120 min), higher c.f.u. values were recovered from biofilms treated with 10 and 20 % (w/v) manuka honey (subinhibitory concentrations) compared with untreated biofilms, with a maximum recovery of 1.2610 6 c.f.u. S. E. Maddocks and others 784 Microbiology To establish whether manuka honey affected binding of S. pyogenes MGAS6180 to the wound-associated proteins fibronectin and fibrinogen, 1 mg of each of these two protein ligands was immobilized to the surface of a microtitre plate. The extent of binding inhibition using a sublethal dose (20 % w/v) of manuka honey was compared with adherence observed in the absence of manuka honey. Binding of S. pyogenes to fibronectin in the presence of 20 % (w/v) manuka honey was reduced by 74 %, which was found to be statistically significant (P50.01); conversely no such reduction in binding was observed for fibrinogen (P50.38) Genes encoding the surface adhesins Sof and SbfI are differentially expressed in response to exposure to a sublethal concentration of manuka honey To determine whether the decreased binding to fibronectin was the result of differential expression of two of the major fibronectin-binding proteins (Sof and SfbI) of S. pyogenes MGAS6180 in response to sublethal concentrations of honey, end point RT-PCR was employed. The PCR products were analysed by densitometry and quantified relative to the New England Biolabs 1 kb quantitative molecular mass Streptococcus pyogenes biofilms and honey http://mic.sgmjournals.org 785 marker (thresholds for detection ¢0.15 nmol). The results confirmed that both sfbI and sof were expressed to a lesser extent in the presence of 20 % (w/v) manuka hone

‘Can you recommend any good STI apps?’ A review of content, accuracy and comprehensiveness of current mobile medical applications for STIs and related genital infections

Objective Seeking sexual health information online is common, and provision of mobile medical applications (apps) for STIs is increasing. Young people, inherently at higher risk of STIs, are avid users of technology, and apps could be appealing sources of information. We undertook a comprehensive review of content and accuracy of apps for people seeking information about STIs. Methods Search of Google Play and iTunes stores using general and specific search terms for apps regarding STIs and genital infections (except HIV), testing, diagnosis and management, 10 September 2014 to 16 September 2014. We assessed eligible apps against (1) 19 modified Health on The Net (HON) Foundation principles; and (2) comprehensiveness and accuracy of information on STIs/genital infections, and their diagnosis and management, compared with corresponding National Health Service STI information webpage content. Results 144/6642 apps were eligible. 57 were excluded after downloading. 87 were analysed. Only 29% of apps met ≥6 HON criteria. Content was highly variable: 34/87 (39%) covered one or two infections; 40 (46%) covered multiple STIs; 5 (6%) focused on accessing STI testing. 13 (15%) were fully, 46 (53%) mostly and 28 (32%) partially accurate. 25 (29%) contained ≥1 piece of potentially harmful information. Apps available on both iOS and Android were more accurate than single-platform apps. Only one app provided fully accurate and comprehensive information on chlamydia. Conclusions Marked variation in content, quality and accuracy of available apps combined with the nearly one-third containing potentially harmful information risks undermining potential benefits of an e-Health approach to sexual health and well-being.The Electronic Self-Testing Instruments for Sexually Transmitted Infection (eSTI2) Consortium is funded under the UKCRC Translational Infection Research (TIR) Initiative supported by the Medical Research Council (Grant Number G0901608) with contributions to the Grant from the Biotechnology and Biological Sciences Research Council, the National Institute for Health Research on behalf of the Department of Health, the Chief Scientist Office of the Scottish Government Health Directorates and the Wellcome Trust

The narrative self, distributed memory, and evocative objects

In this article, I outline various ways in which artifacts are interwoven with autobiographical memory systems and conceptualize what this implies for the self. I first sketch the narrative approach to the self, arguing that who we are as persons is essentially our (unfolding) life story, which, in turn, determines our present beliefs and desires, but also directs our future goals and actions. I then argue that our autobiographical memory is partly anchored in our embodied interactions with an ecology of artifacts in our environment. Lifelogs, photos, videos, journals, diaries, souvenirs, jewelry, books, works of art, and many other meaningful objects trigger and sometimes constitute emotionally-laden autobiographical memories. Autobiographical memory is thus distributed across embodied agents and various environmental structures. To defend this claim, I draw on and integrate distributed cognition theory and empirical research in human-technology interaction. Based on this, I conclude that the self is neither defined by psychological states realized by the brain nor by biological states realized by the organism, but should be seen as a distributed and relational construct

Induction of humoral immune response to multiple recombinant Rhipicephalus appendiculatus antigens and their effect on tick feeding success and pathogen transmission

BACKGROUND: Rhipicephalus appendiculatus is the primary vector of Theileria parva, the etiological agent of East Coast fever (ECF), a devastating disease of cattle in sub-Saharan Africa. We hypothesized that a vaccine targeting tick proteins that are involved in attachment and feeding might affect feeding success and possibly reduce tick-borne transmission of T. parva. Here we report the evaluation of a multivalent vaccine cocktail of tick antigens for their ability to reduce R. appendiculatus feeding success and possibly reduce tick-transmission of T. parva in a natural host-tick-parasite challenge model. METHODS: Cattle were inoculated with a multivalent antigen cocktail containing recombinant tick protective antigen subolesin as well as two additional R. appendiculatus saliva antigens: the cement protein TRP64, and three different histamine binding proteins. The cocktail also contained the T. parva sporozoite antigen p67C. The effect of vaccination on the feeding success of nymphal and adult R. appendiculatus ticks was evaluated together with the effect on transmission of T. parva using a tick challenge model. RESULTS: To our knowledge, this is the first evaluation of the anti-tick effects of these antigens in the natural host-tick-parasite combination. In spite of evidence of strong immune responses to all of the antigens in the cocktail, vaccination with this combination of tick and parasite antigens did not appear to effect tick feeding success or reduce transmission of T. parva. CONCLUSION: The results of this study highlight the importance of early evaluation of anti-tick vaccine candidates in biologically relevant challenge systems using the natural tick-host-parasite combination

Herschel-ATLAS: A Binary HyLIRG Pinpointing a Cluster of Starbursting Protoellipticals

Panchromatic observations of the best candidate hyperluminous infrared galaxies from the widest Herschel extragalactic imaging survey have led to the discovery of at least four intrinsically luminous z = 2.41 galaxies across an 100 kpc region-a cluster of starbursting protoellipticals. Via subarcsecond interferometric imaging we have measured accurate gas and star formation surface densities. The two brightest galaxies span 3 kpc FWHM in submillimeter/radio continuum and CO J = 4-3, and double that in CO J = 1-0. The broad CO line is due partly to the multitude of constituent galaxies and partly to large rotational velocities in two counter-rotating gas disks-a scenario predicted to lead to the most intense starbursts, which will therefore come in pairs. The disks have Mdyn of several 10(sup 11) solar Mass, and gas fractions of 40%. Velocity dispersions are modest so the disks are unstable, potentially on scales commensurate with their radii: these galaxies are undergoing extreme bursts of star formation, not confined to their nuclei, at close to the Eddington limit. Their specific star formation rates place them greater than or approx. equal to 5 above the main sequence, which supposedly comprises large gas disks like these. Their high star formation efficiencies are difficult to reconcile with a simple volumetric star formation law. N-body and dark matter simulations suggest that this system is the progenitor of a B(inary)-type 10(sup 14.6) -solar mass cluster